Within the last decade, the development of improved electron tunneling probes and optical methods has made it possible to detect single molecules in vacuum, solution, the solid-state and on surfaces. In contrast to classical spectroscopy which monitors an average molecular ensemble, single molecule detection (SMD) examines unique events within a population. This ability enables one to examine minor species whose function would normally be lost by inclusion within an average. In solution, single molecules have been detected by monitoring their laser-induced fluorescence (LIF) (Rigler, R. J. Biotech. 1995, 41, 177; Rigler et al. Fluorescence Spectroscopy; Wolfbeis O. S., Ed.; Springer, Berlin, 1992, pp 13-24; Edman et al. Proc. Natl. Acad. Sci. USA 1996, 93, 6710; Goodwin et al. Acc. Chem. Res. 1996, 29, 607; Schmidt et al. Proc. Natl. Acad. Sci. USA 1996, 93, 2926; Keller et al. Appl. Spectroscopy 1996, 50, A12). While the detection limit of LIF is far superior to that of traditional fluorescence spectroscopy, the method cannot directly operate at the single molecule level (i.e., 10-24 M). Amplification to the molecular level can be accomplished by reducing the probed volume to or below a femtoliter. Modern developments in optical imaging now provide confocally-imaged pin holes which when placed in the path of a diffraction limited laser beam provide an illuminated cylinder with a diameter of 400 nm, a length of 2 mm, and a volume of approximately 0.2 fl (1 fl=10-15 1). In this cavity, the concentration of a single molecule now corresponds to 8.3 nM, a value within the capacity of LIF. Using this and other methods for generation of small volumes, single fluorescent molecules have been detected in flowing and static solutions.
Since its discovery in 1974, fluorescence correlation spectroscopy (FCS) has provided new insight into a wide variety of investigations; including diffusion, aggregation, chemical reactions, and conformational analysis. The principle of this method is based on monitoring the fluctuation in fluorescence intensity as molecules diffuse through a specified illuminated cavity. When a molecule or group of molecules pass into a cavity tuned near their absorption maximum, they undergo cycles of excitation and relaxation by emission of a second photon. When these events are recorded in a time-dependent matter, the quanta which belongs to these molecules can be determined through autocorrelation. In 1994, Rigler and Eigen described a method for detecting single rhodamine-labelled DNA molecules, based application of confocal microscopy to FCS. Concurrently, Zare and colleagues devised a similar method for detecting single molecules of YOYO intercalated DNA in real time, without the need for autocorrelation. Since these discoveries, Webb and Gratton have expanded the spectral window to include the UV-region through use of multiple photon excitation. When used in conjunction with techniques for concentrating or separating particles, such as electrical focusing or optical tweezers, the detection limit of this method becomes infinite. To date, FCS has been applied to a number of biophysical investigations, including the study of protein folding, neuroreceptor-binding, the motion of actin-filaments, and membrane dynamics. This investigation describes the first application of single molecule FCS to monitor the interaction between carbohydrates and proteins.
Recently, increased attention has been devoted to gaining a better understanding of the biological significance of carbohydrate-protein recognition, due to the participation of these events in a wide variety of disease related processes including: cellular growth-development, fertilization, metastasis, inflammatory response, as well as bacterial and viral recognition. Low affinity, often with an association constant (Ka) as low as 10.sup.-4 M.sup.-1, has been a major problem facing these investigations. One solution to this problem has appeared through enhancement of the binding by modification of one partner. Several groups have reported dramatically increased affinities of molecules possessing multiple (polyvalent) carbohydrates. The affinity of these ligands and their monomeric counterparts can be determined with techniques such as fluorescence anisotropy or microcalorimetry. The advantage of the latter is that it provides a complete energetic description, including entropic and enthalpic terms.
These method have been used to determine the affinity of sialosides for influenza hemagglutinin, various oligosaccharides for E-selectin, Salmonella trisaccharide epitope for a monoclonal antibody Se 155-4, and numerous C- and O-glycosides for lectins. Although accurate, both methods rely on a comparison between the free and bound state. To date, neither method is capable of specifically detecting molecules in one state nor can either method operate at the single molecule level. One question that becomes important to the understanding of carbohydrate-protein binding events is the role and mechanistic aspects of aggregation before and after binding. Early on, it was recognized that several of the carbohydrate binding proteins exist in aggregated (dimeric, tetrameric, or polymeric) forms. Multivalent ligands, which are already polymeric, are biased towards aggregated forms and therefore do not easily allow one to examine this aggregation. Therefore, discovery of a method which selectively detects only the carbohydrate-bound or free state provides an ideal tool for this type of investigation. Based on the low affinities of these events, the design must incorporate a means to detect at very low concentration, ideally at the single molecule level.
One limitation to the development of confocal FCS and further laser-based fluorometric techniques is the photophysical and spectroscopic properties of the fluorescent molecule or tag. In fluorescence-based single molecule detection, a laser beam tuned near the absorption maximum of the fluorophore is used to initially provide high-lying rotational and vibrational states which then undergo picosecond non-radiative decay to a low-lying singlet state (S1). In doing so, molecules which contain degrees of rotational freedom can adopt more than one singlet state, such as twisted intramolecular charge transfer (TICT) states. These states originate from internal rotation to conformers where the orbitals of one portion of the molecule are oriented orthogonally with the other. Observation of these states was first seen in the fluorescence spectrum of p-N,N-dimethylaminobenzonitrile and soon thereafter attributed internal twisting about the dimethylamino group through analogy to several locked and rotationally restricted derivatives, such as those shown in FIG. 1. Emission from these states is typically sensitive to solvent polarity, low in energy and intensity, and of short lifetime. In addition to formation of TICT states, several fluorophores readily undergo spin-forbidden relaxation from the S1 state to a long-lived triplet state (T1), additionally decreasing in their fluorescence. For single molecule detection, the efficiency of a chromophore is measured with its absorption cross-section (s), its fluorescence quantum yield (Ff) and its photodecomposition.
In addition to photophysical considerations, fluorometric detection of binding requires a substance which undergoes significant modification of its absorption or emission maxima, emission quantum yield and/or excited state lifetime upon binding. Unlike many commonly used fluorophores, (these quantum yields can be compared to other commonly used fluorophores such as fluorescein (0.91 in 0.1 M NaOH) or Rhodamine B (0.70 in is ethanol). Chen, R. F. Anal. Biochem. 1967, 20, 339) the quantum yield of p-(N,N-dimethylamino)-p'-nitro-trans-trans-1,4-diphenyl-1,3-butadiene (NND) decreases upon changing from non-polar to polar solvent systems as seen in the comparison of heptane (Ff=0.097) to methanol (Ff=4.times.10-6). 17 In addition, the absorption and fluorescence maximum of these materials is red shifted by a respective 130 and 109 nanometers over the same interval.
Even more remarkably, the quantum yield of this material increased to 0.14, a 44% improvement over that seen in the most non-polar solvent, when embedded in a phospholipid vesicle.
(Shin et al. J. Phys. Chem. 1988, 92, 2945). This fact clearly shows that restriction of the space for internal rotation results in a dramatic gain of fluorescence.
Similar findings have been seen in the insertion of trans-stilbene into vesicle membranes (Suddabay, et al. J. Am. Chem. Soc. 1985, 107, 5609). In this case, the increased fluorescence quantum yield was attributed to inhibiting photoisomerization, to cis-stilbene, which is known to compete for the singlet excited state with fluorescence. Comparable isomerizations have been seen in p-(N,N-dimethylamino)-p'-nitro-trans-1,2-diphenyl-1,3-ethylene (NNS) (Gorner et al. J. Mol. Struct. 1982, 84, 227; Bent et al. J. Phys. Chem. 1974, 78, 446; Gorner, H.; Schulte-Frohlinde, D. Ber. Bunsen-Ges. Phys. Chem. 1978, 82, 1102). The isomerization yield of this process (Ft-c=0.034 in toluene) as well as the amount of crossing to the triplet state decreased with solvent polarity, suggesting that non-radiative relaxation was the major path back to the ground state in polar solvents. When extensively photolyzed, NND in cyclohexane or toluene reached a photostationary state containing a mixture of the initial trans-trans-isomer (67%) and the corresponding cis-trans isomers. Inhibition of this isomerization may be one factor contributing to the increased fluorescence seen in vesicles.
Alternatively, this fluorescence gain can be explained by restricting the formation of TICT states. Although there is no direct evidence for the presence of TICT states in NND, due to the complicated nature associated with several positions for rotation, the decreased fluorescence and red-shifted absorption upon increasing solvent polarity is comparable with substances known to have TICT states.
Charge transfer labels, such as 5-(dimethylamino)-1-naphthalenesulfonyl or dansyl chloride, have been extensively used for the detection, characterization and localization of carbohydrates, phospholipids, proteins, oligonucleotides as well as numerous other synthetic and natural materials (Seiler et al. Biochem. Anal. 1970, 18, 259). These materials typically experience a shift in their UV/visible absorption and/or fluorescence spectra with respect to the nature of their solvent shell (Reichardt et al., Chem. Rev. 1994, 94, 2319; Kosower et al. J. Am. Chem. Soc. 1971, 93, 2713; Kamlet et al, J.-L. M. Abboud, R. W. Taft, ibid. 1977, 99, 8325).
This effect as well as additional modification of their fluorescence lifetime, amount of intersystem crossing and fluorescence quantum yield have encouraged their use as practical sensors for monitoring the interactions of biologically relevant macromolecules (Weber et al. J. Biochem. 1954, 56, xxxi; Stryer, J. Mol. Biol. 1965, 13, 482; Gally et al. Biochim. Biophys. Acta 1965, 94, 175; Cory et al. J. Am. Chem. Soc. 1968, 90, 1643; Chen et al. Biochem. 1967, 120, 609; Guest et al. Biochem. 1991, 30, 8759). Given by the extent of aromaticity, the dansyl group absorbs light between =190 and 400 nm, limiting its excitation primarily to ultraviolet light (this can be circumvented through use of two or multi-photon excitation, see: Xu et al. J. Opt. Soc. Am. B, 1996, 13, 481).
What is needed is a new class of intramolecular charge-transfer labels which absorb visible and ultraviolet light, display a dramatic solvent sensitivity and can be detected at the single molecule level.